Oral Presentation 51st International Society for the Study of the Lumbar Spine Annual Meeting 2025

Introduction:
Disc degeneration is the leading cause of low back pain and disability. As the disc matrix degrades due to aging or degeneration, immune cells infiltrate, and abnormal nerve growth occurs within the previously aneural and avascular disc, contributing to pain. Understanding the interactions between degenerating discs and neurons is essential to uncovering the underlying mechanisms of discogenic pain, which remains largely elusive. Current animal models fail to replicate the complex multi-organ environment accurately, and existing in vitro models lack the dynamic features necessary to study disc degeneration and innervation. This study presents an “innervation-on-a-chip” microfluidic model designed to mimic nociceptor ingrowth and neuronal activation in response to disc degeneration, allowing real-time analysis of these processes.

Methods:
We developed a microfluidic biochip with separate chambers for discs and neurons connected by 50 µm x 500 µm innervation channels to facilitate directional growth. The Institutional Animal Care and Use Committee approved all procedures. Using C57BL6 mice, we harvested L1-L6 discs with cartilage endplates and lumbar dorsal root ganglia (DRG). Discs and DRGs were dissected, and DRG neurons were isolated, enzymatically digested, and seeded in either the microfluidic biochip or poly-lysine-coated 24-well plates. For co-culture, neurons were seeded in the neuron chamber for 24 hours, after which intact discs or degenerated discs were introduced into the disc chamber. Over 3 days, neurite outgrowth and disc-neuron interactions were observed in real time. Neurite extension was quantified using ImageJ following β-III-tubulin immunostaining, while neuron activation was assessed by dynamic calcium imaging with GCaMP, capturing fluorescence at 480 nm using confocal microscopy. KCl (40 mM) served as a positive control. Data were expressed as mean ± SD and analyzed via one-way ANOVA or t-tests, with statistical significance set at p < 0.05.

Results:
After 3 days of culture, neurite outgrowth was directed toward the degenerative discs, with β-III tubulin staining revealing the longest extensions in the degenerated disc group (Figure 1). Neurons co-cultured with degenerated discs exhibited more branching and complex networks than those in no-disc and intact-disc (ID) groups. Total neurite count and length were highest in the DD group, though average neurite length was shorter in the ID and DD groups due to extensive branching. Calcium imaging indicated a significant Ca2+ influx (2.34 ± 0.05-fold) in neurons exposed to degenerative discs, with KCl treatment inducing a 3.24 ± 0.38-fold increase.

Discussion:
Our study demonstrates the potential of this microfluidic platform to model complex disc-neuron interactions, enhancing our understanding of discogenic pain mechanisms. Results suggest that interactions between degenerative discs and neurons contribute to pain sensitivity and may accelerate disc degeneration. The ongoing investigation into disc-neuro-immune crosstalk within this system may further elucidate pain and degeneration mechanisms, potentially identifying therapeutic targets. By facilitating real-time monitoring of neurite outgrowth and neuron activation, this model overcomes the limitations of traditional methods, offering a valuable tool for developing targeted therapies to alleviate pain and potentially slow disc degeneration.